Structural studies on b-1,4-Galactosyltransferase family

β-1，4-半乳糖基转移酶(Gal-T1)和乳糖合成酶(LS)的结构及新型糖基转移酶的设计：糖链合成缺陷已被证明具有严重的病理后果并导致多种人类疾病。我们的目标是确定半乳糖基转移酶家族成员的结构，这些成员参与合成糖共轭化合物的复杂寡糖结构，并将它们的结构与功能联系起来。半乳糖转移酶亚家族成员Gal-T1在Mn2+存在下将半乳糖(Gal)从UDP-Gal转移到受体GlcNAc。乳清蛋白(LA)是一种乳腺特异的蛋白质，它与Gal-T1酶相互作用形成乳糖合成酶(LS)复合体，从而改变该酶对葡萄糖(GLC)的受体专一性，从而产生乳糖。我们实验室对Gal-T1和LS的结构和生化研究表明，它们类似于一个精致的机械装置，具有两个协调良好的柔性环，包含在Gal-T1催化结构域中。较小的一个含有Trp残基(Trp314)，两侧是甘氨酸残基。较大的一个由345到365个氨基酸残基组成。底物结合后，Trp314侧链移动将糖核苷酸锁定在结合部位，而大环发生构象变化，掩盖了糖核苷酸结合部位，并创建了：1)寡糖结合空腔；2)酶伙伴LA的蛋白质-蛋白质相互作用部位；以及3)金属离子结合部位。在构象变化过程中，His347的定位使得它可以参与与D252VD254序列的Met344和Asp254配位。定点突变和动力学分析表明，Asp254和His347与金属配基结合较强，而Met344配位较弱，可被丝氨酸、谷氨酰胺或丙氨酸取代。D252VD254序列的Asp252不参与Mn2+离子的结合，而是与UDP-Gal的半乳糖部分结合。只有在构象II中，Gal-T1和LA形成LS复合体，使Gal-T1能够选择新的底物葡萄糖。LA将GLC固定在Gal-T1的受体结合部位，从而最大化与GLC的相互作用，从而使其成为LS反应的首选受体。LA与构象II中的Gal-T1的相互作用也稳定了糖-核苷酸-酶复合体，动态地促进了糖的转移，即使是从不太受欢迎的糖核苷酸。 Gal-T家族成员可以表现为特定的凝集素：掩盖糖核苷酸结合位点的构象变化也可以由受体单独诱导。大环中的构象变化在糖核苷酸结合位点上盖上了一个盖子，同时创建了一个延伸的糖结合位点，可以容纳长度为N-糖链的五糖，从而使蛋白质能够作为一种特定的凝集素。Gal-T家族的每个成员在其寡糖结合位点上都显示出序列差异。每一种都以组织特异性的方式表达，并显示出对特定寡糖的偏好。鉴于这些特性，它们可以作为组织特异性凝集素。像LA这样的分子可能与LA结合部位相互作用，LA结合部位也是低聚糖的结合部位，并可能通过竞争低聚糖的结合而作为特异性凝集素抑制剂。 新型糖基转移酶的基于结构的设计：从Gal-T1的晶体结构中获得的信息已被用于设计Gal-T1的突变体，这些突变体对不同的供体和受体底物具有偏好，并使我们能够合成以其他方式在化学上难以合成的寡糖。测定了Gal-T1LA与UDP-GalNAc形成的配合物的晶体结构。决议。结构表明，UDP-GalNAc与Gal-T1的结合与UDP-Gal与Gal-T1的结合非常相似，只是在GalNAc的N-乙酰基与Tyr289的侧链羟基之间形成了额外的氢键。推测这种额外的氢键可以解释Gal-T1的GalNAc转移酶活性较低的原因。对Tyr289突变株的研究证实了这种情况。突变体Y289L在保持Gal-T活性的同时，表现出较高的GalNAc-转移酶活性，接近野生型Gal-T的100%。对Leu289突变体的稳态动力学分析表明，在Gal-T反应中，GlcNAc的Km比野生型增加了近20倍，而供体催化效率(kcat/Ka)对于UDP-Gal和UDP-GalNAc非常相似。本研究表明，在Gal-T家族中，Tyr289/Phe289残基在很大程度上决定了糖供体的特异性。在细胞中，该残基的单点突变有可能改变Gal-T1的糖供体特异性，这可能在细胞过程中具有严重的影响。

Structure of b-1,4-galactosyltransferase (Gal-T1) and Lactose Synthase (LS) and Design of Novel Glycosyltransferases: Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases. Our aim has been to determine the structure of galactosyltransferase family members which are involved in the synthesis of complex oligosaccharide structures of glycoconjugates and correlate their structure with function. Gal-T1, a member of galactosyltransferase sub-family, transfers galactose (Gal) from UDP-Gal to an acceptor GlcNAc in the presence of Mn2+ion. a-Lactalbumin (LA), a mammary gland-specific protein, interacts with Gal-T1 enzyme and forms a lactose synthase (LS) complex that alters the acceptor specificity of the enzyme towards glucose (Glc) to produce lactose. Structural and biochemical investigations from our laboratory on Gal-T1 and LS have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates: 1) the oligosaccharide binding cavity; 2) a protein-protein interacting site for the enzyme's partner, LA; and 3) a metal ion binding site. During conformational change, His347 is positioned in such a way that it can partake in coordinating the Mn2+ion with Met344 and Asp254 of D252VD254 sequence. Site-directed mutagenesis and kinetic analysis, and the crystal structures of the mutants show that Asp254 and His347 strongly bind metal ligand, while Met344 coordinates less strongly and can be substituted by serine, glutamine or alanine. The Asp252 of D252VD254 sequence is not involved in Mn2+ion binding; instead it binds to the galactose moiety of UDP-Gal. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar-nucleotide-enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. Gal-T family members can behave as specific lectins: The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone. The conformational change in the large loop puts a lid - a cover - on the sugar nucleotide binding site, while simultaneously creating an extended sugar binding site that can accommodate an N-glycan, penta-saccharide in length, thus enabling the protein to act as a specific lectin. Each member of the Gal-T family shows sequence variation in its oligosaccharide binding site. Each is expressed in a tissue-specific manner and shows preferences for specific oligosaccharides. Given these properties they can act as tissue-specific lectins. The molecules like LA may interact with the LA binding site, which is also the oligosaccharide binding site, and may act as specific lectin inhibitors by competing for the binding of the oligosaccharide. Structure based design of novel glycosyltransferases: The information derived from the crystal structures of Gal-T1 with less preferred donor or acceptor substrates has been used to design the mutants of Gal-T1 that have preferences for different donor and acceptor substrates and has allowed us to synthesize oligosaccharides that are otherwise chemically difficult to synthesize. The crystal structure of the Gal-T1 LA complex with UDP-GalNAc was determined at 2.1 ? resolution. The structure reveals that the UDP-GalNAc binding to Gal-T1 is quite similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond that is formed between the N-acetyl group of GalNAc moiety with the side chain hydroxyl group of Tyr289. It is reasoned that this additional hydrogen bond would account for the low GalNAc transferase activity of Gal-T1. This condition was substantiated by the studies on the mutants of Tyr289. The mutant Y289L exhibits enhanced GalNAc-transferase activity that approaches 100% of the wild-type Gal-T activity even while retaining its 100% Gal-T activity. The steady state kinetic analyses on the Leu289 mutant indicate that in the Gal-T reaction, the Km for GlcNAc has increased nearly twentyfold compared to wild-type, whereas the donor catalytic efficiency (kcat/KA) is quite similar for both donors, UDP-Gal and UDP-GalNAc. This study demonstrates that in the Gal-T family the Tyr289/Phe289 residue largely determines the sugar donor specificity. In a cell, a single point mutation of this residue has the potential to change the sugar donor specificity of Gal-T1, which may have serious implications in cellular processes.